Our scientific goal is to develop more potent non-viral gene delivery systems to treat genetic diseases of the lung such as cystic fibrosis (CF). Our hypothesis is that non-viral gene delivery systems must surmount multiple barriers in series and that modest improvements at overcoming each barrier can lead to substantial improvements on overall gene expression efficiency. The challenge is to correctly identify the barriers and to devise means to overcome them. We target the lung in this research because the morbidity and mortality associated with CF is due to pulmonary complications from alterations in the gene encoding the CF transmembrane conductance regulator (CFTR) in lung epithelial cells. Furthermore, gene therapy of CF has been documented with plasmid DNA containing the CFTR sequence complexed to cationic lipids (lipoplexes) or cationic polymers (polyplexes) in animals and lipoplexes have been used in human clinical trials to deliver CFTR to the airways. However lipoplex delivery efficiency is low, gene expression is transient, the current complexes have an acute toxicity and little is known concerning their mechanism of action. We propose three approaches to improve gene delivery to the lung by improving the current non-viral systems: Approach 1, involves improved cationic molecules and techniques for compacting DNA. Approach 2, involves improved membrane destabilizing formulations to increase DNA delivery into the cytoplasm of the cell. Approach 3, involves altered and improved surface properties for minimizing toxicity upon injection and increasing gene delivery to target cells. We also will examine ways to improve DNA persistence and distribution in the target cells using plasmids containing nuclear matrix attachment sites, in situ generation of defective viruses from plasmid systems and low molecular weight RNA/DNA hybrids for site-specific CFTR gene correction. The effects of novel molecules and methods of DNA complex assembly will be evaluated in mechanism-based studies using gene expression, radioisotopic, fluorescent, confocal microscopic and electron microscopic techniques to determine: 1. The location, extent and mechanism of DNA release and delivery in cell culture. 2. To determine the mechanism of DNA delivery in vivo via the intravenous and intratracheal routes of administration. A mechanistic understanding of the current systems will help us to significantly improve non-viral DNA delivery by biophysical and biochemical methods and to assess the role of non-viral DNA delivery in the gene therapy treatment of cystic fibrosis.